This invention relates to a material with blood vessel generating properties, uses thereof for encapsulating cells within microcapsules and for forming microspheres, and a method for forming small microspheres and microcapsules.
There are certain situations where it is desirable to vascularize (to form blood vessels in) the tissue of a living animal.
One such situation is in the treatment of heart muscle after an infarction, or heart attack. Depending on the severity of the infarction, some of the heart muscle will be damaged (xe2x80x9cischemicxe2x80x9d) because of the inability of the remaining coronary blood supply to provide enough oxygen and nutrients to it.
Considerable effort has been directed to the use of therapeutic agents such as basic fibroblast growth factor (bFGF) or V-endothelial growth factor (VEGF) (S. Takeshita et al, J. Clin Invest. 1994, 93: 662-670) to generate a collateral blood supply to damaged heart tissue after a period of ischemia and thereby aid in the recovery of the heart after a heart attack. These therapeutic agents are expensive, somewhat unstable and difficult to deliver and have some undesirable side effects. The disadvantage exists that the generation of new blood vessels in ischemic heart muscle is not presently possible without the use of drugs.
Another such situation in which vascularization is desirable is the treatment of peripheral ischemia that may result, for example, as a complication of Diabetes. The microcirculation supplying the peripheral tissue is not able to provide adequate oxygen and nutrients to the legs and feet which results in tissue death, susceptibility to infection, and eventually, amputation.
A further situation is in the treatment of various wounds, including severe burns, or chronic wounds such as bed sores or venous and diabetic ulcers. Chronic wounds are difficult to heal, partly due to an insufficient vascular bed supplying nutrients and healing factors to the wound site.
Another situation is the treatment of the healing interfaces between transplanted and host tissue. For example, the incorporation and survival of cadaverous gum tissue, implanted to replace diseased gum tissue, is hindered by a lack of blood vessels supplying the new tissue with nutrients.
Another situation in which it is desirable to generate new blood vessels is in the field of implantable drug delivery systems, such as microencapsulated animal cells which produce and release a therapeutic agent, such as a biologically active molecule, to the host in which they are implanted.
When any material or device is implanted in the body of an animal (as used herein, terms xe2x80x9canimalxe2x80x9d and xe2x80x9chostxe2x80x9d include humans), the body responds by producing what is termed a foreign body reaction. This involves various leukocytes, particularly macrophages and neutrophils, and typically results in an avascular fibrous capsule that is intended to isolate or xe2x80x9cwall offxe2x80x9d the material or device from the body.
This reaction is appropriate for many situations but not for implantable drug delivery systems, such as implantable microcapsules, where it is preferable to have blood vessels present close to the surface of the material comprising the delivery system. These blood vessels are then able to carry the therapeutic agent to parts of the body where it is needed. An avascular fibrous capsule formed around the drug delivery system acts as a diffusion barrier between the drug delivery system and the body""s blood vessels, preventing or at least slowing the delivery of the therapeutic agent.
The development of fibrogenic tissue around microcapsules has been a persistent and fatal problem with prior art drug delivery systems utilizing microcapsules. In order to survive, implanted cells require free diffusion of nutrients, gases, and waste products while releasing to the host the intended cellular product produced by the microencapsulated cells. However, the formation of scar tissue and lack of vascularization leave the implanted cells effectively cut off from the nutrients necessary for their survival. Therefore, a disadvantage exists that no drug delivery systems utilizing implanted microcapsules are known to date which are vascularized (i.e., create blood vessels in the surrounding tissue in the immediate vicinity of the capsules) when implanted into a host animal.
In order to further promote effective diffusion of essential nutrients, toxic metabolic end-products and cellular products through the microcapsule wall, it is preferred that cells be encapsulated in small microcapsules (defined as microcapsules less than 500 xcexcm in diameter) because of their high surface area and thin walls.
Various techniques have been used for encapsulating mammalian cells in small microcapsules. For example, alginate-polylysine microcapsules having diameters between 250 xcexcm and 350 xcexcm have been prepared with an electrostatic droplet generator, as reported by Sun et al., ASAIO J. 38:125-127 (1992) and Hallxc3xa9 et al., Transplant. Proc. 24:2930-2932 (1992). Alginate-polylysine microcapsules having a diameter of less than 500 xcexcm have been produced with an air-jet droplet generator as reported by Wolters et al., J. Appl. Biomat. 3:281-286 (1992).
Zekorn et al., Acta Diabetol. 29:41-45 (1992), describe a method of encapsulation where pancreatic islets are centrifuged through an alginate solution and a Ba++ containing medium, which yields alginate-coated islets, the xe2x80x9ccapsulesxe2x80x9d having roughly the same size as individual islets and, thereby, effectively eliminating any void volume. There was allegedly no impairment of insulin transport through the alginate coat, although the effect of an additional layer of polylysine was not determined. An interfacial photopolymerization process was reported by Pathak et al., J. Am. Chem. Soc. 114:8311-8312 (1991), for encapsulating pancreatic islets in polyethylene glycol based polymeric microcapsule having a diameter of 500 xcexcm, or for conformal coating as described by Cruise et al., Trans. 19th Ann. Meet. Soc. Biomat. (USA) Abstract 205 (1993). Water insoluble polymers cannot be used as membrane materials by either of these conformal coating/microencapsulation methods.
Encapsulation of mammalian cells in water insoluble synthetic polymer, polyacrylate, for example, by an interfacial precipitation process is described by Sefton and Stevenson, Adv. Polym. Sci. 107:143-197 (1993). Retention of cell viability in vitro and secretion of several bioactive agents have been demonstrated in hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) microcapsules with diameters of approximately 750 xcexcm by Babensee et al., J. Biomed. Mat. Res. 26:1401-1418 (1992), Uludag and Sefton, J. Biomed. Mat. Res. 27:1213-1224 (1993), and Uludag et al., J. Controll. Rel. 24:3-12 (1993). A limitation of this method is that smaller microcapsules cannot be produced due to the relatively low droplet-shearing force as reported by Crooks et al., J. Biomed. Mat. Res. 24:1241-1262, (1990).
However, the disadvantage exists that prior art methods of making microcapsules less than 500 xcexcm in diameter are not particularly efficient. There is still a need for methods which not only produce small diameter microcapsules, but also produce them efficiently, consistently, and with predictable properties.
To at least partially overcome the above disadvantages, the present invention provides an angiogenic material containing a component which causes the generation of blood vessels in surrounding tissue.
Preferably, the angiogenic material comprises a biocompatible polymer and a vascularizing compound, the vascularizing compound preferably being introduced into the biocompatible polymer at the time of polymerization. Because of its biocompatibility, the angiogenic material of the present invention, when implanted in the tissue of a host animal, generates a minimal inflammatory or foreign body reaction in the surrounding tissue. This has the beneficial effect of minimizing the production of inhibitors of growth factors and of angiogenesis more generally. The result is minimization of the avascular fibrous capsule around the angiogenic material and maximization of the capacity of the host to produce the desired blood vessels in the vicinity of the angiogenic material.
Preferably, the vascularizing compound is a polymerizable compound which has anionic character in the final polymer when implanted, for example, polymerizable compounds having ionizable groups such as sulfate, sulfonic acid and carboxylic acid groups. More preferred are polymerizable acids such as acrylic acid, methacrylic acid, crotonic acid, itaconic acid, vinylsulfonic acid and vinylacetic acid, most preferably methacrylic acid. The vascularizing compound, when incorporated in the biocompatible polymer to form the angiogenic material of the present invention, is believed to act as a sink for blood vessel generating growth factors such as VEGF or bFGF. These growth factors are then slowly released by the angiogenic material. As a result of this long release period, blood vessels produced in the vicinity of the angiogenic material are sustained.
In one embodiment of the invention, the angiogenic material is implanted into ischemic heart muscle or in blood vessels close to the heart to stimulate the growth of blood vessels in the ischemic tissue. The angiogenic material may preferably be implanted in the form of a disk, fibre or microsphere, with microsphere being the most preferred form. Preferably, the microsphere has a diameter no greater than about 500 xcexcm, more preferably no greater than about 200 xcexcm, and most preferably from less than about 10 xcexcm to about 50 xcexcm.
In another embodiment, the angiogenic material is implanted into the peripheral ischemic tissue or in blood vessels close to the ischemic tissue to stimulate the growth of blood vessels into the tissue. As with heart tissue, the angiogenic material may preferably be implanted in the form of a disk, fiber or microsphere, with microspheres having the above diameters being the most preferred form.
In another embodiment, the angiogenic material is incorporated into a wound care product applied to the surface of a wound such as a chronic wound or burn. The wound care product may be composed of the angiogenic material or the angiogenic material may be a separate component dispersed in the wound care product in the form of microspheres, disks, or fibers. Wound care products may be wet dressings, dry dressings, occlusive dressings, non-occlusive dressings, wound pastes, or any other product applied to a wound.
In another embodiment, the angiogenic material is placed at the interface between transplanted and host tissue to stimulate growth of host vessels into the transplant to encourage incorporation of the tissue and to supply it with oxygen and nutrients. The angiogenic material may preferably be in the form of small microspheres or fibers, a thin film applied between the new and existing tissue, or at the suture lines.
In another embodiment, the angiogenic material of the present invention is incorporated in the coating of a microcapsule containing cells and/or cell aggregates which, when the microcapsule is implanted in the body of a host, release a cellular product to the host. Microcapsules containing the angiogenic material of the present invention cause generation of blood vessels in tissue surrounding the microcapsule, resulting in more efficient diffusion of cellular products and waste products from the encapsulated cells and better diffusion of nutrients and gases to the encapsulated cells. To further improve efficiency of diffusion, microcapsules of the present invention are preferably no greater than 500 xcexcm in diameter.
The present invention also provides a new method for forming polymeric microspheres and microcapsules encapsulating mammalian cells. This method is particularly useful for formation of microcapsules and microspheres having diameters no greater than about 500 xcexcm. However, it is to be understood that the method of the present invention may be used to prepare microcapsules and microspheres of any suitable dimension.
In the method of the present invention, at least three liquid layers are loaded into a coating chamber to form a density gradient. The three liquid layers are, in order of exposure by the cells, a polymer solution layer containing a coating polymer, a solvent layer and a non-solvent layer. A cell aggregate suspension is introduced at the top or bottom (depending on the density of the aggregates relative to the liquid density gradient) of the coating chamber, centrifugal force is preferably applied, and the conformally coated cell aggregates are removed from the opposite end of the coating chamber.
Preferably, the density gradient comprises five liquid layers, with solvent and non-solvent layers being arranged symmetrically about the polymer solution layer. This allows the density gradient to accommodate cell aggregates with densities either greater than or less than the density gradient layers. Preferably, the liquid layers of the density gradient are, in order from bottom to top: a lower non-solvent layer, a lower solvent layer more dense than the polymer solution, a polymer solution layer containing a coating polymer, an upper solvent layer less dense than the polymer solution layer, and an upper non-solvent layer. The final non-solvent layer into which the aggregates pass is where precipitation of the polymer coating occurs, and is, therefore, also called the precipitation solution layer. Preferably, the upper and lower non-solvent layers are aqueous so that cells enter and exit the coating chamber under aqueous conditions.
Preferred polymers suitable for use in the polymer solution layer include any water insoluble, biocompatible polymer that precipitates in an aqueous environment and is semipermeable after fully precipitating.
In another embodiment of the process of the present invention, microspheres are produced using density gradient layers in a coating chamber as discussed above. However, instead of introducing cell aggregates into the coating chamber, fine gas bubbles are introduced into the polymer solution layer, from which they rise into the upper solvent layer and finally into the upper non-solvent, precipitating layer. Air is the preferred gas because it need not be fed into the coating chamber in a non-solvent suspension and it allows the number of layers in the density gradient to be reduced to three.
The disclosed microcapsules or microspheres can be administered to animals by various means, including implantation, injection, and infusion, via cannulas, catheters, pipette or directly through a needle from a syringe or using forceps or a trocar. When implanted into animals the microcapsules and microspheres according to the present invention will become surrounded with well vascularized tissue.
It is therefore an object of this invention to provide an angiogenic material, which when implanted into tissue of a host animal, promotes growth of blood vessels in the tissue.
It is a further object of this invention to provide microcapsules which promote vascularization of the microcapsules when implanted into a host animal.
It is a further object of this invention to provide microspheres of a material that promotes new blood vessel growth when introduced into heart muscle of a host animal.
It is a further object of this invention to provide an improved method for producing microcapsules and microspheres.
It is a further object of this invention to provide an improved method for producing microcapsules and microspheres having a diameter no greater than 500 xcexcm.
It is a further object of this invention to provide a method of controllably producing small microcapsules with thin walls allowing free diffusion of gases, nutrients and small biologically active molecules.
Accordingly, in one aspect, the present invention provides a method of conformally coating cell aggregates, comprising the following three steps:
a) loading liquid layers into a coating chamber to form a density gradient, wherein the liquid layers comprise in order from bottom to top: a lower non-solvent layer, a lower solvent layer, a polymer solution layer containing a coating polymer, an upper solvent layer, and an upper non-solvent layer;
b) introducing cell aggregates into one of the non-solvent layers of the density gradient; and
c) applying centrifugal force to the coating chamber, such that the cell aggregates pass through each of the liquid layers of the density gradient and thereby become conformally coated by a layer of the coating polymer, wherein said solvent layers contain a solvent capable of dissolving said coating polymer and said coating polymer is substantially insoluble in said non-solvent layers.
In a xe2x80x9csinking cellxe2x80x9d system, the cell aggregates have a density greater than a density of each of the liquid layers of the density gradient, such that the cell aggregates sink downwardly through each of the liquid layers of the density gradient. Conversely, in a xe2x80x9crising cellxe2x80x9d system, the cell aggregates have a density less than a density of each of the liquid layers of the density gradient, such that the cell aggregates rise upwardly through each of the liquid layers of the density gradient.
The order of steps (a) and (b) in the above method may be reversed so that the cell aggregates are introduced into a bottom of the coating chamber before loading of the liquid layers of the density gradient into the coating chamber.
Preferably, the solvent layers contain a solvent which is the same as a solvent in which the coating polymer is dissolved in the polymer solution layer, the non-solvent layers each comprise an aqueous solution and the cell aggregates are introduced into the coating chamber in the form of an aqueous suspension.
Preferably, the conformally coated cell aggregates have a diameter of not greater than about 500 xcexcm.
Preferably, the coating polymer comprises a biocompatible, water-insoluble polymer which precipitates in an aqueous environment and is semi-permeable after precipitation.
Preferably, the coating polymer additionally comprises a vascularizing compound which, when the conformally coated cell aggregates are implanted into tissue of an animal, promotes generation of blood vessels in the immediate vicinity of the implanted conformally coated cell aggregates.
Preferably, said coating polymer is selected from the group comprising polyacrylates, more preferably the polymer solution comprises a solution of hydroxyethyl methacrylate-methyl methacrylate in polyethylene glycol.
In another aspect, the present invention provides a method of producing microspheres, comprising the following steps:
a) loading liquid layers into a coating chamber to form a density gradient, the liquid layers comprising, in order from bottom to top: a polymer solution layer containing a coating polymer, a solvent layer and a non-solvent layer, said solvent layer containing a solvent capable of dissolving said coating polymer and said coating polymer being substantially insoluble in said non-solvent layer;
b) introducing gas bubbles into the polymer solution layer; and
c) allowing the bubbles to rise through each liquid layer of the density gradient so that the bubbles become coated by a layer of the coating polymer to form microspheres.
As in the case of microcapsules, the solvent layer preferably contains a solvent which is the same as a solvent in which the coating polymer is dissolved in the polymer solution layer and the non-solvent layer comprises an aqueous solution.
Preferably, the microspheres have a diameter of not greater than about 200 xcexcm and the coating polymer comprises a biocompatible, water-insoluble polymer which precipitates in an aqueous environment.
Preferably, the coating polymer additionally comprises a vascularizing compound which, when the microspheres are implanted into tissue of an animal, promotes generation of blood vessels in the immediate vicinity of the microspheres.
Preferably, the polymer solution comprises a solution of hydroxyethyl methacrylate-methyl methacrylate in polyethylene glycol; said solvent layer comprises polyethylene glycol; said non-solvent layer comprises distilled water; and said gas bubbles comprise bubbles of air.
In another aspect, the present invention provides a microcapsule comprising: a core containing cellular material selected from one or both of cells and cell aggregates; and a conformal, semi-permeable coating of angiogenic material over the core, the angiogenic material comprising a biocompatible polymer and a vascularizing compound selected from the group comprising polymerizable compounds capable of forming anions, wherein, when implanted in animal tissue, said microcapsule promotes generation of blood vessels in its immediate vicinity and induces minimal or no fibrous capsule formation.
Preferably, the core of the microcapsule additionally comprises a bioactive compound selected from the group comprising growth factors, attachment matrices and immobilization matrices, and the cellular material comprises mammalian cells which produce a bioactive cellular product.
Preferably, the said biocompatible polymer is selected from the group comprising polyacrylates and the vascularization compound comprises a polymerizable compound containing an ionizable group selected from the group comprising sulfates, sulfonic acid groups and carboxyl groups, more preferably acrylic acid, methacrylic acid, crotonic acid, itaconic acid, vinylsulfonic acid and vinylacetic acid, and most preferably methacrylic acid which is incorporated into the biocompatible polymer at the time of polymerization.
Preferably, the microcapsule has a diameter of no more than about 500 xcexcm.
In yet another aspect, the present invention provides an angiogenic material comprising a biocompatible polymer and a vascularizing compound selected from the group comprising polymerizable compounds capable of forming anions; wherein, when implanted in animal tissue, said angiogenic material promotes generation of blood vessels in its immediate vicinity and induces minimal or no fibrous capsule formation.
Preferably, the angiogenic material is in the form of a microsphere having a diameter of less than about 200 xcexcm.
Preferably, the biocompatible polymer is selected from the group comprising polyacrylates and the vascularization compound comprises a polymerizable compound containing an ionizable group selected from the group comprising sulfates, sulfonic acid groups and carboxyl groups, more preferably acrylic acid, methacrylic acid, crotonic acid, itaconic acid, vinylsulfonic acid and vinylacetic acid, and most preferably methacrylic acid which is incorporated into the biocompatible polymer at the time of polymerization.
In yet another aspect, the present invention provides the use of microspheres comprising an angiogenic material for increasing blood flow to ischemic heart muscle, wherein: said microspheres are implanted into the ischemic heart muscle or blood vessels in the immediate vicinity of the ischemic heart muscle, said microspheres having a diameter of from less than about 10 xcexcm to about 50 xcexcm, said angiogenic material comprising a biocompatible polymer and a vascularizing compound selected from the group comprising polymerizable compounds capable of forming anions and which promote the growth of blood vessels, which, when implanted in animal tissue, said angiogenic material promotes generation of blood vessels in its immediate vicinity and induces minimal or no fibrous capsule formation.
Preferably, the vascularization compound comprises a polymerizable compound containing an ionizable group selected from the group comprising sulfates, sulfonic acid groups and carboxyl groups, more preferably acrylic acid, methacrylic acid, crotonic acid, itaconic acid, vinylsulfonic acid and vinylacetic acid, most preferably methacrylic acid which is incorporated into the biocompatible polymer at the time of polymerization.
Preferably, the biocompatible polymer is selected from the group comprising polyacrylates.